Abstract
Protein-disulfide isomerase (PDI) has been proposed to exhibit an "unfoldase" activity against the catalytic A1 subunit of cholera toxin (CT). Unfolding of the CTA1 subunit is thought to displace it from the CT holotoxin and to prepare it for translocation to the cytosol. To date, the unfoldase activity of PDI has not been demonstrated for any substrate other than CTA1. An alternative explanation for the putative unfoldase activity of PDI has been suggested by recent structural studies demonstrating that CTA1 will unfold spontaneously upon its separation from the holotoxin at physiological temperature. Thus, PDI may simply dislodge CTA1 from the CT holotoxin without unfolding the CTA1 subunit. To evaluate the role of PDI in CT disassembly and CTA1 unfolding, we utilized a real-time assay to monitor the PDI-mediated separation of CTA1 from the CT holotoxin and directly examined the impact of PDI binding on CTA1 structure by isotope-edited Fourier transform infrared spectroscopy. Our collective data demonstrate that PDI is required for disassembly of the CT holotoxin but does not unfold the CTA1 subunit, thus uncovering a new mechanism for CTA1 dissociation from its holotoxin.
Highlights
Involved with the single disulfide bridge between CTA1 and CTA2 [3]
In further contrast with the current model of protein-disulfide isomerase (PDI)-toxin interactions where PDI acts as an unfoldase, we found that the PDIinduced shift of CTA1 to a protease-sensitive conformation does not correlate to the disassembly of the cholera toxin (CT) holotoxin
Both antibodies generated an increase in the refractive index of the sensor slide, indicating direct antibody binding to the CT holotoxin
Summary
Materials—His-tagged CTA1, CTA11–168, and CTA11–133 constructs were purified as previously described [26]. The expressed protein was purified in 8 M urea buffer as described previously [26]. Digitonin was used to generate separate membrane and cytosolic fractions as previously described [34] Both cellular fractions were immunoprecipitated with an anti-CTA antibody. Cells were exposed to ice-cold acidic ethanol at 3 h post-transfection and were processed for determination of cAMP levels as described in [34]. An additional set of transfected cells were chased overnight to determine the relative levels of CTA1 expression via metabolic labeling and immunoprecipitation. Values obtained from the cAMP assay were standardized to CTA1 expression levels before expressing the data as percentages of the signal obtained from the parental control cells
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